Composition, method, system and kit for optical electrophysiology

ABSTRACT

The present invention provides a method of optical electrophysiological probing, including: providing a fluorescing chemical probe; contacting a thick portion of tissue with the fluorescing chemical probe to create a thick portion of treated tissue; applying a first range of wavelengths of electromagnetic radiation to the treated portion of tissue; and detecting a plurality of depth-specific emission wavelengths emitted from the thick portion of treated tissue.

RELATED APPLICATION

The present invention is a non-provisional application that claimspriority to U.S. patent application Ser. No. 11/923,282, filed Oct. 24,2007, which corresponds to U.S. Provisional Application No. 60/854,418filed Oct. 24, 2006 and entitled “NEAR-INFRARED STYRYL DYES AND METHODSOF USE IN OPTICAL ELECTROPHYSIOLOGY”. The aforementioned applicationsare incorporated by reference in their entireties.

FUNDING STATEMENT

This invention was made with government support under contractidentifier EB-001963 awarded by the National Institute of Health andcontract identifier HL-071635 and HL-7163501 awarded by the NationalHeart Lung and Blood Institute. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates various embodiments of a composition,method, system, and kit for optical electrophysiology. Morespecifically, the present invention relates to enhanced mapping (and/oroptical imaging) of thick tissue with novel near infrared probes(voltage-sensitive dyes) having high voltage sensitivity, including saidcomposition, method, system, and kit for advanced optical methodsdetailing the electrical activity of an organelle, a cell, a pluralityof cells, a tissue, or an organ, including cardiac tissue andneurological tissue.

2. Related Art

A common method for optically imaging the heart tissue of a subject'sbody is by using microelectrode or patch clamping techniques with branchelectrodes. To utilize branch electrodes, multiple leads are used toinsert needles into various portions of the heart muscle wall. Once theneedles are in place, they record from isolated points inside themuscle. However, branch leads have limitations and drawbacks to theiruse. Branch leads damage portions of the heart muscle, penetrate thetissue (thereby disrupting cross-sectional continuity), and provideuneven measurements due to the size and displacement of the needles fromone another.

These disadvantages have led, in part, to the development of styryl dye,di-4-ANEPPS, which is a voltage-sensitive dye. This dye has been usedfor optically imaging tissue, including cardiac tissue. However, thedi-4-ANEPPS dye has limitations in its use as a voltage probe forcardiac electrophysiology in cells and tissues. Di-4-ANEPPS cannotpenetrate thick tissue at depth; di-4-ANEPPS will only affordmeasurements of optical potentials from only a few hundred micrometersof subsurface layer of tissue. Also, as the excitation wavelength ofdi-4-ANEPPS is the same range in which blood and tissue typicallyabsorb, at 450 to 550 nanometer range in the electromagnetic spectrum,measurements taken with di-4-ANEPPS typically have high scattering andnoise with a low optical resolution, resulting in low image quality evenwith very high light intensity. Hence, a need exists forvoltage-sensitive probes that excite and emit electromagnetic radiationin an electromagnetic range removed from biological interference (i.e.decreased scattering and noise) and provide in depth imaging of thicktissue and/or blood-perfused tissue.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a method of opticalelectrophysiological probing, comprising: providing a fluorescingchemical probe, said fluorescing chemical probe having at least oneexcitation wavelength such that when an electromagnetic radiation of theexcitation wavelength is administered to the fluorescing chemical probe,said probe emits electromagnetic radiation of at least one emissionwavelength, wherein said excitation wavelength and said emissionwavelength are at least about 90 nanometers apart; contacting a thickportion of tissue with said fluorescing chemical probe to create a thickportion of treated tissue; applying a first range of wavelengths ofelectromagnetic radiation to said treated portion of tissue, said firstrange of wavelengths of electromagnetic including said excitationwavelength of said fluorescing chemical probe; and detecting a pluralityof depth-specific emission wavelengths emitted from said thick portionof treated tissue, said plurality of depth-specific emission wavelengthsincremented from a surface of said thick portion of treated tissue to aninner tissue depth from at least about 2.5 millimeters.

A second aspect of the present invention provides an optically mappingcomposition comprising a voltage-sensitive dye of the formula:

wherein R1 includes a first hydrocarbon chain and R2 includes a secondhydrocarbon chain of a length of carbon chains.

A third aspect of the present invention provides a system of in-depth invivo imaging, comprising: a dosage of a fluorescing chemical probe,characteristic in that said probe has an excitation wavelength and anemission wavelength, said excitation wavelength differs by at leastabout 90 nanometers or more from said emission wavelength, said probeconfigured to be biologically compatible to a subject tissue; anillumination source, said illumination source configured to illuminate adosed portion of said subject tissue; a photodetector, configured todetect a plurality of emission wavelength readings from a surface ofsaid subject tissue to at least about 2.5 millimeters of tissue depth;and a computer system, configured to collect and record said pluralityof emission wavelength readings.

A fourth aspect of the present invention provides: An optical probingkit for tissue, comprising: a fluorescing probe quantity, saidfluorescing probe quantity configured to emit an electromagneticradiation emission at about 600 to about 1000 nanometers; an instructionfor administering said fluorescing probe quantity; and a deliverymember; said delivery member configured to deliver said fluorescingprobe quantity into a portion of tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described in detail below with reference to thedrawings in which:

FIG. 1 depicts a flowchart of an example of an embodiment of the methodof the present invention;

FIG. 2 depicts a flowchart of another example of an embodiment of themethod of the present invention;

FIG. 3 depicts a flowchart of still another example of an embodiment ofthe method of the present invention;

FIG. 4 depicts a flowchart of still yet another example of an embodimentof the method of the present invention;

FIG. 5 depicts a flowchart of further an example of an embodiment of themethod of the present invention;

FIG. 6 depicts an example of an embodiment of the system of the presentinvention;

FIG. 7 depicts another example of an embodiment of the system of thepresent invention;

FIG. 8 depicts an example of an embodiment of the computer system of anembodiment of the system of the present invention;

FIG. 9 depicts an example of an embodiment of the kit of the presentinvention;

FIG. 10 depicts another example of an embodiment of the kit of thepresent invention;

FIG. 11 depicts an illustration of examples of an embodiment of thecomposition of matter of the present invention;

FIG. 12 depicts an illustration of examples of an embodiment offluorescing chemical probes of the present invention;

FIG. 13 depicts an illustration of a chemical synthesis for an exemplaryembodiment of a composition of matter of the present invention;

FIG. 14 depicts another illustration of a chemical synthesis for anexemplary embodiment of a composition of matter of the presentinvention;

FIG. 15 depicts an illustration of an example of a fluorescing chemicalprobe of an embodiment of the present invention;

FIG. 16 depicts another illustration of an example of a fluorescingchemical probe of an embodiment of the present invention;

FIG. 17A depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 17B depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 18A depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 18B depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 19A depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 19B depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 20A depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 20B depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention;

FIG. 21A depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention; and

FIG. 21B depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention; and

FIG. 22A depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention; and

FIG. 22B depicts an illustration of experimental data acquired withrespect to the various fluorescing chemical probes with respect to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method, composition, system, and kitfor optical electrophysiology of in situ tissue, and may further includecardiology and neurology applications. Voltage sensitive dyes, alsoreferred to as voltage sensitive probes herein, may be used tofacilitate diagnostic tests, modeling programs, imaging procedures, aswell as optical probing kits. Further to the present invention, thefollowing paragraphs provide additional discussion and disclosure of thevarious embodiments of the present invention, as well as variousexamples of those embodiments. Although the various embodiments of themethod, composition of matter, system, and kit of the present inventionwill be discussed and disclosed in detail inter alia, it should beunderstood by those skilled in the art that the applications referencedhere and the various examples and embodiments of the present inventionmay be used in the heart, in the brain, or throughout the body of anorganism, including warm blooded animals, for mapping of the electricalpotential of a single cell, various portions of tissue, and/or organs.

It would be advantageous for the voltage-sensitive probes to have fastresponse time, high voltage sensitivity, a wide range of dye-loadingconstants, low washout time constants, and/or a low photo bleachingeffect in vivo. Further, it would be advantageous for thevoltage-sensitive probes which may be used to measure the membranepotentials across the inner membranes of individual mitochondria withina single living cell, investigate spiral and scroll waves inmillimeter-thick layers or across whole ventricular wall (at or above 10mm thickness), reconstruct three-dimensional scroll waves in a modelingprogram, probe deep tissues, map blood-perfused tissue, and provide moreefficient double-dye (Ca/V_(m)) optical mapping. Also, thevoltage-sensitive probe may have one or more advantageous properties forperforming these tasks at high efficiency and effectiveness, includingefficient loading, low toxicity, and voltage-sensitive efficiency.

Incorporated by reference by their entirety are the followingpublications, including: Matiukas, A., B. G. Mitrea, A. M. Pertsov, J.P. Wuskell, M. D. Wei, J. Watras, A. C. Millard, and L. M. Loew. 2006.New near-infrared optical probes of cardiac electrical activity. Am JPhysiol Heart Circ Physiol. 290:H2633-43.; Matiukas, A., B. G. Mitrea,M. Qin, A. M. Pertsov, A. G. Shvedko, M. D. Warren, A. V. Zaitsev, J. P.Wuskell, M.-d. Wei, J. Watras, and L. M. Loew. 2007. Near InfraredVoltage Sensitive Fluorescent Dyes Optimized for Optical Mapping inBlood-Perfused Myocardium. Heart rhythm: the official journal of theHeart Rhythm Society.; Wuskell, J. P., D. Boudreau, M. D. Wei, L. Jin,R. Engl, R. Chebolu, A. Bullen, K. D. Hoffacker, J. Kerimo, L. B. Cohen,M. R. Zochowski, and L. M. Loew. 2006. Synthesis, spectra, delivery andpotentiometric responses of new styryl dyes with extended spectralranges. J Neurosci Methods. 151:200-15.; Teisseyre, T. Z., A. C.Millard, P. Yan, J. P. Wuskell, M.-d. Wei, A. Lewis, and L. M. Loew.2007. Non-linear Optical Potentiometric Dyes Optimized for Imaging with1064 nm Light. J. Biomed. Opt. 12:044001. (Published 8/07); and Zhou,W.-L., Y. Ping, J. P. Wuskell, L. M. Loew, and S. D. Antic. 2007.Intracellular long wavelength voltage-sensitive dyes for studying thedynamics of action potentials in axons and thin dendrites. J. Neurosci.Methods. 164:225-239.

An example of an embodiment of the present invention includes a methodof optical electrophysiological probing 100. The method may be shown anddescribed, for example, in FIG. 1 through FIG. 5. The method of opticalelectrophysiological probing 100 may comprise, for example, providing afluorescing chemical probe 110; contacting a thick portion of tissuewith said fluorescing chemical probe to create a thick portion oftreated tissue 120; applying a first range of wavelengths ofelectromagnetic radiation to said treated portion of tissue 130, saidfirst range of wavelengths of electromagnetic including said excitationwavelength of said fluorescing chemical probe; and detecting a pluralityof depth-specific emission wavelengths emitted from said thick portionof treated tissue 140, said plurality of depth-specific emissionwavelengths incremented from a surface of said thick portion of treatedtissue to an inner tissue depth at least about 2.5 millimeters.

Providing a fluorescing chemical probe 110 may include a fluorescingchemical probe 200. The fluorescing chemical probe 200 may have both atleast an excitation wavelength 210 and at least an emission wavelength220. That is, the fluorescing chemical probe 200 may have more than oneexcitation wavelength 210 or emission wavelength 220. The respectiveemission wavelengths and excitation wavelengths may be present in, forexample, a band of wavelengths. Also, the excitation wavelength 210 ofelectromagnetic radiation may be, for example, past the range ofwavelengths in which biological tissue absorbs, between about 480 to 550nm.

When the fluorescing chemical probe 200 may be illuminated withelectromagnetic radiation within its range or band of excitationwavelength 210, the fluorescing chemical probe 200 may absorb theelectromagnetic radiation such that the radiation places the fluorescingchemical probe 200 in an excited state. While in the excited state, thefluorescing chemical probe 200 may emit electromagnetic radiation at anemission wavelength 220. For example, an excitation wavelength may be anelectromagnetic radiation wavelength from at least about 630 nm. Anemission wavelength may be an electromagnetic radiation wavelength fromat least about 700 to about 900 nm.

The fluorescing chemical probe 200 may be designed such that theexcitation wavelength 210 of electromagnetic radiation and the emissionwavelength 220 of electromagnetic radiation may be about 90 nanometersapart. In such a manner, ascertaining the difference betweenelectromagnetic radiation administered to the fluorescing chemical probe200 and the electromagnetic radiation emitted from the excitedfluorescing chemical probe 200 molecules may be easily differentiated,filtered, and measured for voltage intensity.

In reference to the fluorescing chemical probe 200, this may refer to avoltage-sensitive dye, for example, a near infrared voltage sensitivedye. Also, fluorescing chemical probe 200 may both excite from and emitradiation in the electromagnetic spectrum that may exceed or have awavelength greater than 600 nanometers. The operable range, whichincludes both the excitation and emission ranges of the dyes, may be inthe visible red, in the near infrared, and/or in the infrared, ranging,for example, from about 600 nm to about 1000 nm.

The fluorescing chemical probes 200 that may be utilized in the methodof optical electrophysiological probing 100 may comprise, for example,novel derivatives of one or more dyes in the styryl class of compounds.The dyes may measure and respond to fast electrophysiological processesthat occur in cell membranes. Some examples of these processes includeionic currents (currents of ion channels) that may in effect buildaction potential of a cell. Another example of a fastelectrophysiological process may include an external electric field thatmay depolarize or polarize the cell membrane of a cell as in the case ofelectric defibrillation of the heart. For example, the probes 200 mayinclude various dyes from the JPW class of compounds synthesizedoriginally by Joeseph P. Wuskell and Leslie Loew at the University ofConnecticut Health Center. Some examples of fluorescing chemical probes200 are depicted, for example, in FIG. 11 through FIG. 12. The synthesisof these two classes of dye compounds may be shown and described, forexample, in FIG. 13 through FIG. 16.

Referring to FIG. 11, the first class of dyes, may be referred to asClass A, including, JPW 3067, JPW 5020, JPW 5034, and generic class A,respectively. All three fluorescing chemical probes JPW 3067, JPW 5020,and JPW 5034 have the same chromophore, but may differ by the length ofhydrocarbon chains. Molecular weights of three class A examples,including JPW 3067, JPW 5020, and JPW 5034 are 659.59 g/mol, 743.74g/mol, and 855.95 g/mol, respectively. The JPW 3067, JPW 5020, and JPW5034 may each have an absorbance spectra which may reveal several maximathat have variable amplitudes under different pH or solvent conditionsand may represent different configurational isomers. The probes mayabsorb out to around 800 nm. The class A probes comprise absorption andemission peaks that occur at wavelengths 150 nm longer than those ofdi-4ANEPPS.

Referring to FIG. 12, the second class of dyes may be referred to asClass B, including JPW 6003 (Di-4-ANBDQPQ) and JPW 6033 (Di-4-ANBDQBS),with Di-4-ANEPPS for comparison. Both dyes in class B have the samechromophore, but may differ by quaternary ammonium and butylsulfonatepolar groups. The molecular weights two class B example dyes, JPW 6003and JPW 6033, are 695.63 g/mol, and 570.73 g/mol, respectively. Theabsorption maxima (excitation wavelength) for JPW 6003 in ethanoloccurred at 603 nm. For the dye JPW 6033, absorption maxima (excitationwavelength) may shift 20-40 nm to shorter wavelengths, but absorbance istwo to three times higher than JPW 6003. Though the long wavelengthabsorbance peaks of class B fluorescing chemical probes may range from561 nm to 603 nm in ethanol, and 526 nm to 539 nm in a model membranemade of multilamellar lipid vesicles (MLV), in practical applications,both fluorescing chemical probes 200 may be efficiently excited over amuch broader range (500-700 nm), allowing one to optimize excitation forthe dye in different tissues, including blood perfused tissue. Whenclass B probes may be excited at 560 nm in ethanol, both probes emitover the range 700-900 nm, though the fluorescence of JPW 6033 inethanol is much lower. In MLVs, the fluorescence of JPW 6033 to 520 nmlight is considerably higher, with emission over the range 600-800 nm,and peak fluorescence slightly greater than that of JPW6003. Inmyocardium, both class A voltage sensitive dyes and class B voltagesensitive dyes may emit fluoresce in the form of emission wavelength inthe near infrared region 700-900 nm.

Various figures support one or more characteristics of the fluorescingchemical probes 200 of class B, including, for example, JPW 6003 and JPW6033. FIG. 17 illustrates the Dye absorbances in ethanol (FIG. 17A) andmultilamellar lipid vesicles (FIG. 17B). FIG. 18 depicts dye emissionspectra in ethanol (FIG. 18A), and multilamellar lipid vesicles (FIG.18B). FIG. 19 depicts the wavelength dependent relative transmittedlight (FIG. 19A) and fluorescence (FIG. 19B) responses on thehemispherical lipid bilayer (HLB) for 100 mV voltage steps. In FIG. 19B,the fluorescence emission was collected through a >715 nm cutoff filter.

FIG. 20 depicts the fluorescence response of the class B fluorescingchemical probe 200, JPW 6003, in various cardiac tissues. In FIG. 20A,the very bottom trace shows the performance of di-4-ANEPPS in rat forcomparison with the class B dye JPW 6003. Further traces from bottom totop correspond to rat, guinea pig, pig, and transillumination mode inpig, respectively. The measurements were taken of tissue perfused byTyrode's solution. In FIG. 20B, the bottom or lower trace shows theperformance of di-4-ANEPPS, while the upper trace shows the performanceof the class B JPW 6003 probe in a blood-perfused pig tissue.

FIG. 21 shows fluorescing chemical probe 200 loading and washoutdynamics in a subject rat. FIG. 21A and FIG. 21B each show the dynamicsfor the normalized total fluorescence, and voltage-sensitivefluorescence ΔF/F, respectively. The dynamics of the dye di-4-ANNEPPS isshown in both FIG. 21A and FIG. 21B for comparison. Also, the error barsin the FIG. 21A show the level of data variation. All curves in FIGS.21A and 21B are averages of 3 to 5 experiments.

FIG. 22 shows dye (fluorescing chemical probe 200) loading and washoutdynamics in blood-perfused pig heart. The three curves show dynamics ofthe normalized OP amplitude, normalized total fluorescence, andvoltage-sensitive fluorescence ΔF/F (also referred to as delta F/F).FIG. 22 A depicts dye loaded in Tyrode solution, which later wasswitched to blood at the time moment shown by the arrow in FIG. 22A.FIG. 22B depicts the dynamics of the dye loading through directinjection into blood flow, which is shown for comparison to FIG. 22A.

The JPW dyes of class A and class B, or the functional equivalentsthereof, with various R groups on the generic JPW′ class A and class Bmolecules may be used in conjunction with the method of opticalelectrophysiological probing 100. Though the dyes presented hereininclude structural similarities, it is herein noted that one or moredyes may likewise be used in the method, provided the dyes share or aresimilar to the desirable characteristics, features, and functions of thedyes disclosed herein in a manner such that they may be accorded intothe embodiment of the present method 100.

For example, the JPW class B voltage sensitive dyes of the presentinvention may be classified according to some of the various propertiesthat they exhibit with respect to dye imaging. For example, the JPW 6003dye may show delta F/F values of approximately 20% in a cell membrane ofa target thick target tissue (thickness at about 10 mm), in which deltaF/F is essentially the dye's fluorescence emission value or fluorescingintensity. In blood-perfused cardiac tissue, the dye may exhibitcharacteristics of 12% for delta F/F values at about 10 mm thickness.

The method of optical electrophysiological probing 100 may furthercomprise the step of contacting a thick portion of tissue with saidfluorescing chemical probe to create a thick portion of treated tissue120. Thick tissue may refer to tissue that is approximately a fewmillimeters thick to tens of millimeters thick. For example, a portionof thick tissue 70 may be from about 2.0 mm thick to about 20 mm thick,such as about 15 mm thick for example.

Contacting a thick portion of tissue, as used herein, may refer to oneor more of the methods of applying the fluorescing chemical probe 200 toa portion of thick tissue without penetrating the portion of tissue thatis to be optically probed by the method 100. Contacting may be selectedfrom the group consisting of: close-proximity injecting, diffusing,circulating, topically applying, and combinations thereof as may be usedin the art. Therefore, an example of contacting may include injectingthe fluorescing chemical probe 200 into a portion of tissue from whichit will diffuse, circulate, or otherwise transfer to the portion oftissue referred to as the treated portion of tissue.

As another example, contacting a thick portion of tissue may includeapplying the dye to the interior or exterior of the tissue or organ tobe probed, such that through cohesion, adhesion, and diffusion, thefluorescing chemical probe 200 may be passively transferred through theportion of tissue to a given depth, or to the other side of the tissuethereby completely treating a cross-section of tissue.

As still another example, contacting may include injecting or otherwisedistributing the dye into the circulatory system of a subject that inturn circulates the dye to the appropriate portion of thick tissue.Likewise, as yet still another example, contacting may include injectingthe dye into a space, including, for example, a chamber of a heart suchthat the dye is in close proximity to the walls and/or surface of thetissue and may be diffused therethrough. Also, it should be noted thatwhen the organism's body may be opened with surgical applications, thedye may be administered topically either with or without additionaladditives, including a medicament to aid in treating a medical symptomor diagnosis, or a diagnostic agent to aid in the assay or otherdetermination of a heart or tissue condition or illness.

A thick portion of treated tissue 75 may refer to the area of tissuethat has been contacted with the fluorescing chemical probe 200 andthereafter substantially retains a concentration of the fluorescingchemical probe 200 for at least a minimal amount of time prior tointernalization (wash out). A thick tissue may be, for example, about 2millimeters thick, about 5 millimeters thick, about 10 millimetersthick, or about 20 millimeters thick.

It should be noted that the step of contacting a thick portion of tissue70 with said fluorescing chemical probe 200 to create a thick portion oftreated tissue 75 may be facilitated by combining the fluorescingchemical probe 200 with one or more diluents, adjuvants, solvents, ordelivery agents. Alternatively, the dye may be in a pharmacologicallyacceptable salt form to facilitate delivery into a targeted thickportion of tissue 70 and biocompatibility.

For example, the solid fluorescing chemical probe 200 may be dissolvedin dimethylsulfoxide (DMSO) to make a solution of dye. For example, thefluorescing chemical probe 200 stock solution may comprise a 10-50microMolar stock solution, which may in turn be kept under argon purge,vacuum sealed, stored at low temperature, or kept frozen until ready foruse in the exemplary embodiment of the method 100. The fluorescingchemical probe 200 DMSO stock solution may be injected, imbibed, orotherwise contacted to the thick portion of tissue. For example, the dyesolution for injection may be made by dissolving a portion of stocksolution into a portion of Ringer's solution or Lock-Ringer's Solution(for example, several milliliters of Ringer solution). For example,Lock-Ringer's solution may comprise 140 millimoles/liter (mmol/l) NaCl,5.6 mmol/l KCl, 1.0 mmol/l MgCl₂, 5.0 mmol/l HEPES, 10.0 mmol/l Glucose,2.0 mmol/l NaH₂PO₄ and 2.2 mmol/l CaCl₂.

Also, to facilitate contacting of the fluorescing chemical probe 200 toa portion of thick tissue, Pluronic F-127 may be added to the finalconcentration of stock solution and Ringer's or Locke-Ringer's solution.Pluronic F-127 is an FDA approved loading agent that may typically beused for loading dyes like di-4-ANEPPS. For example, Pluronic F-127 maybe added to the final concentration at about 0.1% to about 1.0%, morespecifically, about 0.2 to about 0.5%. As other examples of diluents,ethanol may be utilized. Alternatively another loading agent similar toF-127, may be utilized. Alternatively, cyclodextrins may be utilized,including for example gamma cyclodextrin. While cyclodextrins may beused to encapsulate the dyes and increase solubility, the encapsulationitself may lower the effective delivery of the dye to the subjecttissue.

Contacting a thick portion of tissue with said fluorescing chemicalprobe 200 may be done with various dye concentrations. For example, thedose of JPW 6003 or JPW 6033 dye may be on the order of about 1 to10,000 nanomoles. As another example, the dose of JPW 6003 and 6033 maycomprise about 10 to about 100 micromoles. As still yet another example,the fluorescing chemical probe 200 may be contacted to a portion ofthick tissue 70 at a concentration of about 100 nanomoles per 1 gram ofcardiac tissue.

After the step of contacting with a fluorescing chemical probe 200 toyield a treated portion of thick tissue 120, the method of opticalelectrical physiological probing 100 may comprise applying a first rangeof wavelengths of electromagnetic radiation to said treated portion oftissue 130.

Applying electromagnetic radiation 230 having a first range ofwavelengths may refer to administering through a light source includinga visible light source, laser, or other means to bring the first rangeof wavelengths 230 into contact with the treated portion of thick tissue75 such that the electromagnetic radiation having the first range ofwavelengths 230 may be absorbed by the tissue and by the fluorescingchemical probe 200 that is diffused therein.

The light source may be, for example, a light source that may generateand administer red visible light. As another example, a laser lightsource may generate and administer near infrared electromagneticradiation, infrared electromagnetic radiation, or far infraredelectromagnetic radiation onto the thick portion of treated tissue suchthat the tissue and dye may absorb the radiation applied. For example,light from a tungsten lamp may be sent through a monochromator andfocused onto the surface of a thick portion of treated tissue 75 or amodel of a hemispherical lipid bilayer stained with a fluorescingchemical probe 200. In vivo, the tissue, such as heart tissue, may havevoltage potential changes across the cells, in milliVolt detectableranges. In a model system, a train of voltage steps may be incorporated,for example ±50 mV voltage steps may be applied to the membrane at afrequency of 40 Hz. Then, the modulation of the detected light signal(plurality of emitted wavelengths) may be measured with a lock-inamplifier.

It should be noted that due to the advantageous properties class A andclass B fluorescing chemical probes, and their excitation ranges ofwavelengths which are significantly shifted from the band at whichtissue and blood absorb, visible red light, near infrared light, andinfrared light may be utilized with this embodiment of the presentinvention. As such, the level or intensity of light or electromagneticradiation which may be required to acquire an acceptable image and/orreading is much lower than the light intensity needed with thedi-4-ANEPPS dye of the same class. That is, the intensity of excitationlight may be, on average, seven to ten orders of magnitude lower withvarious embodiments of the present invention. This may be, for example,because there is much less scattering and noise with the excitation andemission wavelengths or wavelength bands as both are shifted from thebands at which biological tissue and materials absorb light. As aresult, the light source, illumination source, and/or laser source mayoperation at a much lower intensity and still yield a much higherquality image.

The first range of wavelengths of electromagnetic radiation 230 may beselected from those wavelengths or like band of wavelengths which mayhave a correlation to a wavelength, more than one wavelength, or a bandof wavelengths at which the excitation wavelength 222 of fluorescingchemical probe 200, and likewise, the treated portion of tissue 75 maybecome excited and emit at least a detectable quantity of fluorescingelectromagnetic radiation. This may be at a maximum excitationwavelength, or a notable and measurable excitation wavelength, such thatcost, ease of administration (e.g. biocompatibility, retention time, andfluorescing intensity) are maximized with respect to economics,efficiency, effectiveness, and practicality. That is, because the probestypically have excitation bands including a range of wavelengths, it ispossible to select a lower cost or available light source that mayexcite the dyes within its excitation band, though not at its wavelengthof maximum absorption. As such, the first range of wavelengths 230 ofelectromagnetic including said of said fluorescing chemical probe 200may be measurable and distinguishable (part of the excitation band) andtherefore practical for use with this embodiment of the present method100, although not exactly at the lambda max values for the fluorescingchemical probe 200.

After the step of applying a first range of wavelengths 130, the methodof optical electrophysiological probing 100 comprises detecting aplurality of depth-specific emission wavelengths emitted from said thickportion of treated tissue, said plurality of depth-specific emissionwavelengths incremented from a surface of said thick portion of treatedtissue to an inner tissue depth of at least about 2.5 millimeters 140.

Detecting may be done with various types of detectors, including fiberoptic devices, photodiode arrays, digital cameras, and/or visualinspection. For example, the use of detectors, including, for example,high-speed video cameras or photodiode arrays may yield high spatial andtemporal resolution recordings of electrical activity in the layers ofcardiac tissue. Also, these types of detectors may permitvisualization/observation and recording of, for example, complexstructures including spiral waves.

The detector may be set up such that it is on the same side of thetissue as the light source, such that the detection is done through areflection of light technique. Also, the detector may be set up on theopposite side of the tissue from the light source, such that thedetection is done through a transillumination of light technique. Also,there may be more than one detector set up such that both reflection andtransillumination methods simultaneously collect data. That is, intransillumination mode, the excitation source (illumination device) andan emission detector (detector) may be on substantially opposing sidesof a thick portion of treated tissue. One or the other method may bepreferred based on differing scientific and experimental variables.

The plurality of depth-specific emission wavelengths 240 emitted fromsaid thick portion of treated tissue 75 may refer to more than oneemission wavelength 240 which is emitted from the thick portion oftreated tissue 75. As the fluorescing chemical probe 200 is comprised ofa plurality of molecules, after the molecules diffuse into the portionof thick tissue 70 to yield a treated portion of thick tissue, moleculesof the fluorescing chemical probe 200 at different levels of thicknessmay likewise be excited by the application of a first range ofwavelengths 230 including at least a portion of the excitationwavelength 210. In such a manner, upon excitation of a treated portionof thick tissue, a plurality of depth-specific emission wavelengths 75will be emitted.

Then, one may review the emissions in an incremented form such that, forexample, there are available detailed emission wavelengths from an areaof the treated portion in a cross-section, ranging from the surface 71of said thick portion of treated tissue 75 to an inner tissue depth 76.The inner depth 76 may range from about a few microns from the surface71 of the thick portion of tissue to about 15 millimeters. Theincrementation may be on the molecular level, cell-by-cell,layer-by-layer of tissue, one or more microns, one or more millimeters,or any other predetermined thickness determination method known to thosein the art. The inner depth may be measured as a displacement from thesurface of the treated tissue portion. For example, the inner depth of athick portion of treated tissue may be at least about 2 millimeters. Asanother example, the inner depth of a thick tissue portion of treatedtissue may be about 15 or 20 millimeters from the surface of the treatedportion of tissue.

The method of optical electrophysiological probing 100 may furthercomprise the step of recording data relating to the plurality ofdepth-specific emission wavelengths 150. Once the plurality ofdepth-specific emission wavelengths 240 have been detected during thedetecting step 140, the data therefrom (results) thereof may be recordedto create a permanent record of the readings for use in research,diagnostics, or storage on to one or more media storage devices whichmay be computer readable medium, or otherwise accessible with one ormore electronic devices. The various types of computer readable mediaand electronic devices that may be used in conjunction with thisexemplary embodiment of the method 100 of the present invention may bedisclosed and discussed infra with respect to the discussion of anaspect of the present invention detailing a system.

The method of optical electrophysiological probing 100 may furthercomprise analyzing the plurality of depth-specific emitted wavelengthsto determine an at least one physical parameter 160. Analyzing mayinclude correlating, calculating, measuring, diagnosing, characterizing,deriving, or otherwise determining at least one physical parameter. Theanalyzing step may be done, for example, by a clinician, a physician, aresearcher, a technician, or other person, termed a “user”, of knowledgeand skill in the art to correlating, calculating, measuring, diagnosing,characterizing, deriving, or otherwise determining at least one physicalparameter. Also, the user may complete the analysis step in combinationwith a computer system. Alternatively, the computer system maycorrelate, calculate, measure, diagnose, characterize, derive, orotherwise determine at least one physical parameter completely withproper programming and/or software, which may then be either stored,presented, or stored and presented to a user. The user may then base adiagnosis, treatment plan, or one or more variables on the at least onephysical parameter.

The at least one physical parameter may refer to a single physicalparameter variable, which may be measured once, or a plurality of timesover a length of time or cycle, or a plurality of various physicalparameters which may be measured either once or a plurality of timesover a length of time or a cycle. The at least one physical parametermay be, as previously disclosed, one of more differing variables of asubject's thick portion of treated tissue. For example, one or more ofthe physical parameters may be selected from the group consistingessentially of: a tissue health, an electrical potential, a thickness,an emission wavelength intensity, a two-dimensional model, athree-dimensional model, a computational model, a cross-sectional model,a disease characteristic, a diagnostic condition, a symptom, a bloodperfusion, circulation, effectiveness, a molecular event, a diseaseprogression, a high-resolution cross-sectional image, and combinationsthereof.

A tissue health may refer to how well a tissue, for example, a cardiactissue, conducts voltage and exhibits membrane potentials across one ormore areas, how well a dye is absorbed into the tissue, and if thetissue has one or more defects in it which may be readily ascertained bythe absorption or non-absorption of the fluorescing chemical probe 200.An electrical potential may refer to the ability of a tissue, includingfor example a heart tissue, to readily conduct voltage that is needed inorder to create an impulse through the cardiac tissue to initiate theheart muscle to contract and cause the heart to beat. The fluorescingchemical probe 200 may be essentially a voltage-sensitive dye such thatthe probe will only fluoresce at points where it has absorbed into themembrane of a cell that is exhibits a change in electrical potential. Athickness may refer to understanding the measurement of a cross sectionof a treated portion of thick tissue at various points in order tounderstand how the various cells of the tissue may be aligned near oneanother, including location of tissue bundles, blood vessels feeding thecardiac tissue with oxygen, etc. The emission wavelength intensity mayrefer to the level of fluoresce that may be detectable or otherwiseobserved from a thick portion of treated tissue which has been excitedwith a first wavelength of electromagnetic radiation including theexcitation wavelength, thereby exhibiting a fluorescing emissionwavelength. The intensity may further refer to a comparative analysisbetween one area's intensity and another area's intensity, where areamay refer to various points of a treated portion of thick tissue that isexhibiting a detectable or observable amount of emission wavelength of,for example, a fluorescing-type character. A computational model mayinclude, for example a two-dimensional model, a three-dimensional model,a cross-sectional model of a portion of thick treated tissue or theentire organ, for example a heart. A disease characteristic may include,for example, low intensity of fluorescing emission wavelength in one ormore areas. A diagnostic condition may include, for example, a screeningof a subject's treated portion of tissue to determine if one or morevariables exist. A symptom may refer to a physical parameter that may beindicative of a disease, a condition, or suggest a need to perform oneor more diagnostics on one or more thick treated portions of tissue.Other physical parameters may include, as previously disclosed a bloodperfusion, circulation, effectiveness of heart as a pump, a molecularevent, including an electrical impulse or change in membrane potential,a disease progression, a high-resolution cross-sectional image, andcombinations thereof. It should also be noted that the physicalparameter may be a function of the subject (organisms), age, weight,height, body surface area, or relative condition of the subject's heart.

The method of optical electrophysiological probing 100 may furthercomprise administering a second range of wavelengths of electromagneticradiation to said thick treated portion of tissue after a predeterminedperiod of time 170. The second range of wavelengths of electromagneticradiation 250 may be the excitation wavelength of the fluorescingoptical probe 200, or a band of wavelengths in which the intensity offluorescing from the treated portion of thick tissue may be observableor detectable. Administering the second wavelength 170 may be equivalentto applying a first wavelength 130, yet at a different predeterminedtime of administration. For example, there may be a predeterminedpassage of time between applying the first wavelength and administeringthe second range of wavelengths, for example, such that the twowavelengths define a treatment plan, screening plan, or diagnostic of athick portion of treated heart tissue. Also, the method of opticalelectrophysiological probing may be configured such that the first rangeof wavelengths of electromagnetic radiation and a second range ofwavelengths of electromagnetic radiation are both of differentintensities of electromagnetic radiation or different beam sizes.Further, the first range of wavelengths of electromagnetic radiation anda second range of wavelengths of electromagnetic radiation may comprisepartially overlapping or completely different ranges of electromagneticradiation. This may be helpful, for example, in procedures in whichdifferent classes of dyes may be co-administered, wherein the dyes mayhave a slightly overlapping or different range of wavelengths in whichan excitation wavelength or range of wavelengths lies. Also, one or moreof the steps of the method of optical electrophysiological probing maybe reiterated once, twice, or more.

Another aspect of the present invention may be a system of in-depth invivo imaging. The system of in depth in vivo imaging 300 may comprise: adosage of a styryl probe, an illumination source, and a photodetector.The system of in depth in vivo imaging 300 may also comprise a computersystem. The elements and features of an example of an embodiment of thesystem of in depth in vivo imaging 300 may be discussed below, withreference to FIG. 6 through FIG. 8.

The system of in depth in vivo imaging 300 may comprise a dosage 310 ofa fluorescing chemical probe 200. The styryl probe may be, for example,class A or class B voltage-sensitive dyes of one of the formulaspreviously discussed, or otherwise similar to the aforementioned dyeswith respect to structure of operability in the defined and describedsystem. The fluorescing chemical probes 310 may be characteristic inthat said probe has an excitation wavelength 210 and an emissionwavelength 220, said excitation wavelength 210 differs by at least about90 nanometers or more from said emission wavelength 220. This differencein wavelengths of the excitation and emission wavelengths may be morecommonly referred to as a Stokes shift.

A Stoke's shift may refer to the difference (in wavelength units)between positions of the band maxima of the excitation (absorption) andemission spectra in, for example, fluorescing species which may beconfigured to be biologically compatible to a subject tissue. A largeStokes shift (i.e., the difference between the excitation and emissionwavelengths) may make them particularly convenient for microscopy,because a large Stokes shift eases the exclusion of scattered andreflected light and the filtering away of background autofluorescence.Also, the fluorescing chemical probe 200 may have a low level oftoxicity, referred to generally as biocompatibility.

The illumination source 320 of the system may refer to a light source322 or laser source 324 which may provide electromagnetic radiation ofor including the excitation wavelength 210 of a range of wavelengths forthe fluorescing chemical probe 200. The illumination source 320 may befurther configured to illuminate a dosed portion of subject tissue 78.

The photodetector 330 of the system 300 may refer to a photodiode array320, a charge coupled device camera 330, high-speed camera, or similardevice. The photodetector 330 may be configured to detect a plurality ofemission wavelengths 245. The emission wavelengths 245 may be readingsfrom a surface of said subject tissue 72 to a predetermined tissue depth76 of at least about 2.5 millimeters of tissue depth. The readings maybe recovered and sorted in an incremented fashion.

The system 300 may further comprise a computer system 340. The computersystem 340 may be configured to collect and record said plurality ofemission wavelength 245 from said surface of subject tissue 72 to thepredetermined tissue depth 76. The computer system 340 may be disclosedand described with reference to FIG. 8 and the following paragraphs.

The computer system 340 may comprise, for example, a processor and acomputer readable memory unit coupled to the processor, said memory unitcontaining instructions that when executed by the processor implement amethod. The method may comprise the method for opticalelectrophysiological probing for tissue 100, including heart tissue.Alternatively, the method may comprise a method of screening a subjectfor a heart disease or condition, a method of diagnosing said physicalsymptoms as one or more heart diseases or conditions, a method ofmonitoring drug delivery and efficacy to tissue, including heart tissue,and/or a method for monitoring a user's heart effectiveness uponmodification of one or more variables, including oxygen level, bloodpressure, ineffective tissue (no electrical potential across a cell'smembrane), etc.

An example of an embodiment of the computer system 340 of the presentinvention may provide a process for supporting computer infrastructure,said process comprising providing at least one support service for atleast one of creating, integrating, hosting, maintaining, and deployingcomputer-readable code in a computing system, wherein the code incombination with the computing system is capable of performing a method.For example, the computer system may be capable of performing an opticalelectrophysiological probing method of 100 of the present invention,similar to the previous exemplary embodiment disclosed.

The computer system 340 comprises a processor 341, an input device 342coupled to the processor 341, an output device 343 coupled to theprocessor 341, and memory devices 344 and 345 each coupled to theprocessor 341.

Input device 342 may be, inter alia, a keyboard, a mouse, a keypad, atouchscreen, a voice recognition device, a sensor, a network interfacecard (NIC), a Voice/video over Internet Protocol (VOIP) adapter, awireless adapter, a telephone adapter, a dedicated circuit adapter, etc.

The output device 343 may be, inter alia, a printer, a plotter, acomputer screen, a magnetic tape, a removable hard disk, a floppy disk,a NIC, a VOIP adapter, a wireless adapter, a telephone adapter, adedicated circuit adapter, an audio and/or visual signal generator, alight emitting diode (LED), etc.

The memory devices 344 and 345 may be, inter alia, a cache, a dynamicrandom access memory (DRAM), a read-only memory (ROM), a hard disk, afloppy disk, a magnetic tape, an optical storage such as a compact disc(CD) or a digital video disc (DVD), etc. The memory device 345 includescomputer code 347 which is a computer program that comprisescomputer-executable instructions.

The computer code 347 includes, inter alia, an algorithm used formapping the electrical potential of organelles, cells, and tissuesaccording to the present invention. The processor 341 executes thecomputer code 347. The memory device 344 includes input data 346. Theinput data 346 includes input required by the computer code 347. Theoutput device 343 displays output from the computer code 347. Either orboth memory devices 344 and 345 (or one or more additional memorydevices not shown in FIG. 8) may be used as a computer usable medium (ora computer readable medium or a program storage device) having acomputer readable program embodied therein and/or having other datastored therein, wherein the computer readable program comprises thecomputer code 347.

Generally, a computer program product (or, alternatively, an article ofmanufacture) of the computer system 340 may comprise said computerusable medium (or said program storage device).

Any of the components of the present invention may be deployed, managed,serviced, etc. by a service provider that offers to deploy or integratecomputing infrastructure with respect to a process for system forelectrophysiological probing of thick tissue of the present invention.Thus, the present invention discloses a process for supporting computerinfrastructure, comprising integrating, hosting, maintaining anddeploying computer-readable code into a computing system (e.g.,computing system 340), wherein the code in combination with thecomputing system is capable of performing one or more methods of orrelated to the present invention.

While FIG. 8 shows the computer system 340 as a particular configurationof hardware and software, any configuration of hardware and software, aswould be known to a person of ordinary skill in the art, may be utilizedfor the purposes stated supra in conjunction with the particularcomputer system 340 of FIG. 8. For example, the memory devices 344 and345 may be portions of a single memory device rather than separatememory devices.

The system 300 may further comprise an administration member 370 foradministering said dosage of said fluorescing chemical probe 200 to asubject tissue 70. The administration member 370 may comprise, forexample, a transdermal patch, an intravenous line, a syringe, a bolousinjection, a solution, a pill casing, and/or a topical application. Theadministration member 370 may be selected such that the administrationmeans 370 conforms to the characteristics of the fluorescing chemicalprobe 200 and/or any solubilizing or delivery agents thereof tofacilitate effective and efficient administration of the fluorescing dyeprobe 200 to a subject tissue 70.

A still yet another aspect of the present invention includes an opticalprobing kit for tissue 400. The optical probing kit 400 may be shown anddescribed with reference to the paragraphs that follow as well as FIG. 9through FIG. 10. The optical kit for tissue 400 may comprise, forexample, a fluorescing chemical composition 410; an instruction 420; anda delivery member 430.

The fluorescing chemical composition 410 may refer to a fluorescingchemical probe 200 selected from one of those previously discussed whichmay be in combination with a delivery agent, solubilizing agent, orother material. This combination may facilitate dosing,biocompatibility, stability, solubility into a host organism or subjecttissue, or otherwise create an ease of packaging, handling, andadministration. The fluorescing chemical composition 410 may beconfigured such that the fluorescing chemical composition 410 may emitan electromagnetic radiation emission wavelength band from at leastabout 600 to about 1000 nanometers.

The optical probing kit 400 may further comprise an instruction 420. Theinstruction 420 may comprise a direction, dosing amount, or instructionfor administering said fluorescing chemical composition 410 to a user ora subject tissue 70. The instruction 420 may be, for example, a relevantmaterial safety data sheet, an instruction for mixing the dosagesolution, a detailed listing of the side effects of taking thefluorescing chemical composition 410, the physical parameters that thefluorescing probe may measure, the treatment plan for administering thefluorescing chemical probe, the instruction for safely disposing of thenon-utilized or left over fluorescing chemical compound 410, poisoncontrol phone numbers, manufacturer information, etc. That is, theinstruction 420 may be one or more pieces of information which a user orsubject may find helpful in administering the fluorescing chemicalcomposition 410 to a thick portion of tissue 70 to be treated.

The optical probing kit 400 may further comprise a delivery member 430.The delivery member 430 may be configured to deliver said fluorescingchemical composition 410 into a portion of tissue. That is, the deliverymember may comprise an apparatus by which the fluorescing chemicalcomposition is injected, topically applied, internally applied,ingested, or otherwise circulated throughout the subject or organism toa subject thick portion of tissue to be treated by the kit 400. Forexample, the delivery member may comprise a syringe, a pipette, atransdermal patch, a pill, or combinations thereof.

The optical probing kit 400 may further comprise a computer programproduct 440. The computer program product 440, may comprise, forexample, a computer usable medium having a computer readable programcode embodied therein. The computer readable program code may, forcontain, for example, instructions that, when executed by a processor ofa computer system, implement a method for manipulating the data acquiredthrough administration and illumination of said fluorescing chemicalcomposition 410 in a user or subject's tissue, including heart tissue

The computer software program 440 of an example of an embodiment of thepresent invention may allow a user, including a clinician, a technician,a researcher, a student, a physician, and/or a professor to collect,manipulate, and analyze one or more readings that may be acquired withthe kit. The computer software program may allow a user to create one ormore of the following, including: a three dimensional model of at leasta portion of a user's tissue, a two dimensional image of at least aportion of a user's tissue, a cross-sectional image of a user's thickportion of tissue, or a physical parameter of a subject or thick portionof treated tissue, said physical parameter a single measurement, aplurality of measurements, or plurality of measurements with respect totime, drug administration, progressing symptom, etc.

As still yet another aspect of the present invention comprises anoptical mapping and signal measuring composition 250. Theelectrophysiological measuring composition 250, may comprise a voltagesensitive dye of the following formula:

That is, the electrophysiological measuring composition 250 may compriseone or more voltage sensitive dyes selected from the group consisting ofclass A JPW dyes. That is, based on the JPW class A generic structureabove, R1 and R2 may be selected from many various chemical functionalgroups. For example, R1 and/or R2 may be selected from varioushydrocarbon groups, including alkyl groups, alkenyl, alkynyl, benzyl,and phenyl groups. Also, the R1 and R2 may be made up of one or moreother functional groups, though, for example, hydrocarbon groupsattached at the tertiary amine sites may generally increase solubilityin a biological host or model. Therefore, R1 and R2 may be eachindependently selected from the groups consisting of methyl, ethyl,butyl, propyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl alkanefunctional groups. Alkyl groups of a chain length may associate to acell membrane very easily, thereby increasing the dyes solubility andoverall sensitivity. Alternatively, although a thiophene is used as ajunction linker in the chromophore, other heterocyclic compounds mayalso be utilized, for example, furan. In the class A compound of thepresent invention, R1 and R2 may be the same functional group. Forexample, R1 and R2 may be methyl groups, as is the case in, for example,JPW 3067. As another example, R1 and R2 may be butyl groups, as is thecase in, for example, JPW 5020. As still another example, R1 and R2 maybe octyl groups, as is the case in, for example JPW 5034. Alternatively,R1 and R2 may be different groups, with different sizes, sterics, and orcharacteristics such that R1 and R2 may collectively allow the compound250 of the present invention to associate and or otherwise affiliatewith one or more cells.

Further to the embodiment of the electrophysiological measuringcomposition 250, the composition may further comprise a delivery agent260. The delivery agent 260 may be at least one of a physiologicallyacceptable adjuvant, solvent, or diluents. For example, the deliveryagent may comprise pluronic F-127, cyclodextrin, gamma cyclodextrin,dimenthylsulfoxide (DMSO), ethanol, or combinations thereof.

As the probes are used in each aspect of the various embodiments andexamples of the present invention, as a preliminary matter, the relevantsynthesis and characterization of the dyes is variation of polar groupscan be used to optimize properties such as dye penetration andpersistence, or voltage-sensitivity. It should be noted that the dyesensitivity is very high in both class A and class B materials. Furtherdye sensitivity is dependent upon the signal to noise level which may beachieved in measurement. The lower range of the probes for a processedsignal may be 0.1 mV. This may correspond to a signal to noise ratio(processed) of 1000. The upper limit or range in which the probes maydetect voltage is limited to the amount of voltage that may typicallybreak down the dye molecule, which may typically be around a few toseveral volts. The other issue which may affect voltage-sensitive dyesensitivity is linearity between measured voltage and optical signal,which, in the present invention, the linearity may hold up underconditions of 500 mV.

The novel probes provide, for example, improved performance in terms ofloading, internalization, washout and membrane-voltage-sensitivity. Thismay in turn enable various new experiments to be completed in the areaof deep tissue probing of blood perfused tissue, which includes theexamples and embodiments of the various methods and systems disclosedand discussed herein. Also, this may enable more efficient double-dyeoptical mapping (Ca/Vsub.m). Also, the particularly effectivefluorescing chemical probes may be utilized in the field of cardiacelectrophysiology, where the probes may provide sub-millisecond temporalresolution, high fluorescing, stable staining, low photobleaching, andlow toxicity. That is, the probes may be used in conjunction withvarious embodiments of the method, system, and kit of the presentinvention with respect to almost any type of tissue or even individualcells which exhibits changes in membrane potential. Besides applicationsin, for example, cardiac cells and tissues, additional applications maylie in the area of the brain and individual neurons.

The method, system, composition of matter, and kit may be used in orderto effectively understand and collect the time-varying optical signalproportional to the membrane electrical potential in cells, tissues, ororgans. Alternatively, it is possible to collect time-independent signalwhich may provide anatomical features of the tissue. Though anatomicalfeature collection may be completed at or near the surface of a subjecttissue, deeper probing of the changes in membrane potential may bemeasured both in close proximity to the tissue surface as well as indeeper layers. For example, the various embodiments of the probes,method, system, and kit of the present may be used in order to collectoptical signals in deep tissue, up to 5 or even 7 millimeters in depth.For even deeper readings, including up to and over 10 mm thickness, itmay be necessary to independently collect signal (fluorescence emissionwavelengths) from both sides of a subject tissue. For this reason, morethan one detector may be placed on either side of the subject tissue torecord data accordingly.

Another application of utilizing the voltage sensitive dyes, includingthe fluorescing chemical probes may include, for example, accessingmembrane potentials in cell population measurements with aspectrofluorometer or determine spatial patterns of voltagedistributions associated with tissues, individual cells, or organelles.

Another favorable feature of the styryl dyes is their high fluorescencequantum efficiency when bound to membranes but negligible fluorescencein aqueous solution; thus only stained cells contribute to thefluorescence signal even if the experimental protocol does not permitwashing away the staining solution. The existing repertoire of styrylpotentiometric dyes has varying solubility, lipid avidity, tissuepenetrability, and ionic charge that allows them to be customized forspecific types of experimental demands.

The applications of the embodiments of the present invention extend toinvestigating spiral and scroll waves in millimeter thick layers oracross whole ventricular wall (thickness up to ˜10 mm), with the finalgoal being to reconstruct 3D scroll waves. As the fluorescing chemicalprobes provide lower light scattering and less background fluorescence,the probes may be utilized in investigating the spiral and scroll wavesin millimeter thick layers across, for example, and entire ventricularwall. The entire ventricle wall may be, for example, up to about 10 mmthick to about 15 or 20 mm thick.

The transillumination method, as previously discussed, as well as narrowilluminating beam scanning and transmitted light recording may be usedin combination with the embodiments of the present invention withrespect to deeper imaging. Thus, in real biomedical applications, thenovel probes, system, kit, and method of the present invention may showmany advantages over the existing blue-red repertoire of voltagesensitive dyes. Some of these advantages may include, for example,greater depth of imaging, compatibility with blood perfused samples, andprolonged retention. Also, though the dyes may be structurally similar,differences in the dyes may cause differences including, for example,solubility in lipids, tissue penetrability, and ionic charge. Thesevarious factors may provide the opportunity to customize dyes for use inone or more types of environments or specific applications.Additionally, optical mapping techniques for blood-perfused cardiactissues may allow the study of many specific features of an in vivoheart, including ventricular fibrillation (VF) baseline.

Experimental Data, Including Examples:

JPW 6003, JPW 6033:

Methods: Dye Spectra and Sensitivity Measurement in Model Membranes.

Voltage-dependent spectra in model membranes were measured by avoltage-clamped hemispherical lipid bilayer (HLB) apparatus modified fornear infrared fluorescence detection. In this experiment, light from atungsten lamp is sent through a monochromator and then focused onto thebottom of the HLB, stained with dye from the external aqueous bathingsolution. The monochromator is then scanned over the wavelength range ofinterest while a train of ±50 mV voltage steps are applied to themembrane at a frequency of 40 Hz and the modulation of the detectedlight signal is measured with a lockin amplifier. The transmitted lightsignal is collected at 180° from the incident light. Fluorescence iscollected at 90° via fiber optic light guide through cutoff filters at715 nm.

Cardiac Tissue Preparation and Staining Methods

The dye testing experiments were performed on typical model animals:mice (n=6), rat (n=11), guinea pig (n=6), pig (n=13). All experimentalprotocols conformed to the Guide for the Care and Use of LaboratoryAnimals and were approved by the Committee for the Humane Use of Animalof the SUNY Upstate Medical University. Mice (C57BL/6, 20-25 g) wereinjected with ketamine (200 mg/kg IP), rats (Sprague Dawley, 300-400 gfemale) and guinea pigs were injected with heparin (550 U/100 g), andeuthanized by sodium phentobarbital (1 ml/100 g for rats and 0.75 ml/100g for guinea pigs). The heart was then immediately excised and placed inice-cold cardioplegia solution (CPS) composed of (in mmol/l) 280Glucose, 13.44 KCl, 12.6 NaHCO₃, 34 Mannitol. After removal ofextraneous tissues, the aorta was cannulated and Langendorff perfusionwas started with a standard oxygenated Tyrode's solution (composed of(in mmol/l) 130 NaCl, 24 NaHCO₃, 1.2 NaH₂PO₄, 1.0 MgCl₂, 5.6 Glucose,4.0 KCl, 1.8 CaCl₂; buffered to a pH of 7.4) at 80 mm Hg and 36° C.Young pigs (15-20 kg,) were heparinized (500 IU, IV) and subsequentlyanesthetized with sodium phentobarbital (35 mg/kg IV). The heart wasrapidly removed and Langendorff-perfused with cold (4° C.) CPS. Theright free ventricular wall was quickly excised, and the right coronaryartery was cannulated. Non-perfused tissue was removed, leaving apreparation of typically 5×5 cm and a thickness of 8 mm. The preparationwas stretched on a plastic frame and perfused with a standard oxygenatedTyrode's solution at 80 mm Hg and 36° C. Whole cannulated heart (mouse,rat or guinea pig) or stretched pig tissue was put into specialtransparent chamber and superfused with the same solution at a rate of30-40 ml/min. Perfusion and superfusion temperatures were continuouslymonitored and kept at 36±1° C. by using two sets of glass heating coilsand heated-refrigerated circulators. Electrodes were sutured to wholehearts or inserted into pig tissue to monitor ECG. All preparations werecontinuously paced at the frequency 6.7 Hz, 5 Hz, 3.3 Hz and 2 Hz formouse, rat, guinea pig, and pig, respectively.

Blood-perfused Langendorff preparation of pig heart was implemented.Briefly, approximately 1.5 liters of modified Tyrode's solution (37° C.,composition in mmol/L: Na⁺, 157; K⁺, 4.7; Ca²⁺, 1.5; Mg²⁺, 0.7, H₂PO₄⁻0.5, Cl⁻137.6, HCO₃ ⁻28.0, glucose 11.0, dextran 4% and insulin 10 U)was infused in an external jugular vein, and blood-Tyrode's mixture wascollected at equal rate from a carotid artery. The heart was perfusedwith blood-Tyrode mixture in a Langendorff apparatus. The blood-Tyrodemixture will be oxygenated (CO₂ 5%/0₂ 95%), heated (37° C.) and filteredusing a Liliput hollow fiber oxygenator (COBE Cardiovascular, Arvada,Co). Collector tubes were inserted into the right and left ventricles tocollect all the blood coming out of the coronary sinus and Thebesianveins. Thereafter, the heart was “sealed” to ensure that all the outflowof perfused blood is collected for recirculation. The heart was placedin a warm bath with heated water-jacketed transparent glass wall and wassuperfused with an oxygenated, heated Tyrode solution. After a 20-30 minstabilization verified by a vigorous contraction and normal sinusrhythm, the excitation-contraction uncoupler diacetyl-monoxime (DAM) wasadded to the perfusate (15 mmol/l) to stop contractions. ECG wasmonitored throughout the experiment to ensure stable capture of theventricles by the pacing electrode. In each experiment, 300 microL ofthe stock solution (18.8 mg/ml) of JPW6003 were mixed with 1 ml of thestock solution of Pluronic-127 (2 g in 10 ml DMSO) and dissolved in 30ml of Tyrode's solution. For di-4-ANEPPS staining 40 microL of the stocksolution (5 mg in 1 ml DMSO) was diluted in 40 ml Tyrode's solution tothe final concentration of 10 □M. The dyes were injected directly intothe aortic cannula during uninterrupted aortic perfusion.

After a 20-30 min stabilization period during which the perfusion flowand ECG was monitored, the excitation-contraction uncouplerdiacetyl-monoxime (DAM) was added to the perfusate (15 mmol/l) to stopcontractions of cardiac tissue. After a 20 minute equilibration, thepreparation was stained by injecting voltage-sensitive dye solution intothe perfusion flow (a bolus injection near cannula).

Solid dyes were dissolved in DMSO to make 10-50 mM stock solution thatwas kept frozen. The dye solution for injection was made by dissolvingthe required amount of the stock solution into 1-5 ml Ringer solution(composed of (in mmol/l) 140 NaCl, 5.6 KCl, 1.0 MgCl₂, 5.0 HEPES, 10.0Glucose, 2.0 NaH₂PO₄ and 2.2 CaCl₂). To facilitate loading of JPW6003,pluronic F-127 was added at the final concentration of 0.2-0.5%. Variousdye doses (1-1000 nmol) were administered, and optimal doses andconcentrations were found by trial and error. 10-100 μM were found to beoptimal dye concentrations, and 100 nmol per 1 g of cardiac tissueoptimal doses of dye JPW6003. We used only up to 30% of these doses inthe case of pig preparations because of much higher total dyeconsumption. It is recommended that dye loading (perfusion) be performedat lowered temperatures (32-36° C.) because of the tendency of theconcentrated dye solution to evoke arrhythmias (ventricular tachycardiaor fibrillation) immediately or a few minutes after dye injection at 36°C. The normal excitation wave propagation in cardiac tissue was verifiedby injecting a small amount of di-4-ANEPPS (3-10 nmol) both at thebeginning and end of each experiment (using appropriateexcitation/emission filters to prevent crosstalk between the dyes, asdescribed below).

Optical Action Potential Recording

The optical setup for testing the new dyes was similar to the opticalmapping setup described previously. Briefly, a collimated beam providedby a 250 W tungsten halogen lamp uniformly illuminated the epicardialsurface of the preparation. The light was heat filtered and then passedthrough an excitation filter (520+/−40 nm for di-4-ANEPPS; 650+/−20 nmfor the new styryl dyes). Optical action potentials (OP) were recordedwith a cooled fast CCD camera (Little Jo by SciMeasure) with a ComputarH1212FI lens (focal length 12 mm (6 mm for mouse heart), 1:1.2 apertureratio; diameter 28 mm; CBC Corp), located at about 100 mm (20 mm formouse heart) from the sample. Lenses of both the light source and camerawere adjusted to illuminate and image a 25 mm in diameter area at thecenter of the preparation; hence spatial resolution in the images was0.31 mm/pixel. The fluorescent light emitted by the voltage sensitivedye was isolated from excitation using 640±50 nm band pass filter fordi-4-ANEPPS, or a 720 nm long-pass filter for the styryl dyes. Thecamera was located either on the same side of preparation forepifluorescence mode recording or at the opposite side fortransillumination mode recording. To reduce cardiac tissue motionartifacts DAM was used as described above; whole rat or guinea pighearts were additionally gently pressed against stretched nylon mesh (ona single side opposite to both light source and camera so as not toaffect the amount of collected fluorescence). These measures in mostcases completely eliminated motion artifacts.

Blood-perfused pig heart imaging was done in a similar way. Briefly, thedye was excited with a 400-watt tungsten-halogen lamp (excitationfilter, 650+/−20 nm) and the fluorescence was collected above 715 nmusing a Computar objective lens (HG1208FCS_HSP, F0.8, focal length 12mm,) and externally cooled CCD camera (Dalsa CA-D1-0128T, Ontario,Canada) at the frame rate of 400-800 fps and 12-bit resolution.

The acquired images were offline processed to improve their quality. Thebackground autofluorescence (endogenous fluorescence recorded under thesame conditions before dye injection) was subtracted from each frame toobtain the voltage-dependent optical signal. To allow temporal alignmentand subsequent averaging of successive paced action potentials, thetrigger for the pacing stimulus was recorded as a single pixel in themovie frames. The alignment error was no more than one-half frame (˜0.5ms). We used ensemble averaging to improve signal to noise ratio (i.e.,averaging the OP signals from 10-20 sequential recordings). To furtherreduce noise, the OP signal was low-pass filtered in both time (with 3-7point triangular window, depending on the camera frame rate), andspatial domain (with pyramidal 5×5 kernel). The effective temporal andspatial resolutions were 1.76-3.33 ms and 0.78 mm, respectively. Dyeloading and washout dynamics were assessed from averaged sets of OPrecordings (3-5 specimens). To facilitate comparison, fluorescence andOP signals were normalized to their peak values (defined as thedifference between maximum and minimum values) in some figures.

For the dye loading dynamics, the time to reach peak values ofbackground fluorescence (membrane voltage independent component of thedye molecules fluorescence), OP, and ΔF/F were determined by simplevisual inspection of the dynamic plots. For the dye washout dynamics,analogous half-times of the same parameters were determined.

Absorbance spectra were measured in ethanol (panel A), and a modelmembrane made of multilamellar lipid vesicles (MLV) composed of eggphosphatidyl choline (panel B). The absorbance spectra reveal singlemaxima that represent a high level of the dye purity and absence ofdifferent configurational isomers. The absorption maxima for JPW6003 inethanol occurred at 603 nm. For the dye JPW 6033 all absorption maximaare shifted 20-40 nm to shorter wavelengths, but absorbance is 2-3 timeshigher. Though the long wavelength absorbance peaks range from 561 nm to603 nm in ethanol, and 526 nm to 539 nm in MLV, in practicalapplications both dyes can be efficiently excited over a much broaderrange (500-700 nm), allowing one to optimize excitation for the dye indifferent tissues, including blood perfused tissue.

Improved Voltage Sensitivity and S/N Ratio JPW 6003, JPW 6033

The new dyes were found to perform efficiently in various cardiactissues and provide OP 50-100% greater to those of di-4-ANEPPS. The mostefficient dye, JPW6003 showed delta F/F=20% in a model membrane, and18.7% in pig tissue. Also this dye performed very efficiently in thickcardiac tissue (˜10 mm): in transillumination mode (when an excitationsource and an emission detector are on opposite sides of tissue) JPW6003showed delta F/F=20%. JPW 6033 showed slightly lower values, at least inpart due to the use of excitation and emission filter sets that werefound optimal for the dye JPW6003. The absorption and emission peaks forJPW6033 are shifted by 20-30 nm relative to JPW6003, so optimization offilter sets for JPW6033 may yield further improvement in itsperformance.

It should be noted that raw optical signals are quite noisy: signal tonoise ratio of raw signals were 20-40. To obtain good quality it isnecessary to apply standard noise reduction procedures such asaccumulation of the periodic optical signal and spatiotemporalfiltering. Though 10-20 sequential Ops were typically accumulated, ifvery detailed dynamics are not of primary interest, it is possible toaccumulate an even larger number of OPs. It is well known that the noiselevel decreases as the square root of the number of accumulated samples.Spatiotemporal filtering allows further noise reduction by filtering outCCD camera noise, and possible vibrations of the tissue surface in thesuperfusion flow. These procedures allowed to increase signal to noiseratio to 500-1000.

Optical Mapping of a Blood-Perfused Tissue

The dye JPW6003 performed very efficiently, exhibiting rapid loading,high voltage sensitivity, slow washout, and slow internalization. Thekinetics of dye loading in blood-perfused hearts was quite differentfrom those perfused with Tyrode's solution. When the dyed was injectedduring recirculated-blood-perfusion there was a very slow increase intotal fluorescence and OP (˜1 hour) whereas delta F/F remainedpractically constant (at the level of 5-6%) after 2 min of injection.When the dye was loaded during Tyrode's perfusion (FIG. 7A), the initialloading was fast and delta F/F value has high (up to 12%), but return toblood perfusion caused a large gradual decrease in total fluorescence,OP, and deltaF/F. Interestingly, the steady-state level of deltaF/Fduring blood perfusion was similar (˜5%) regardless whether the dye wasinjected during Tyrode's perfusion with subsequent blood perfusion or itwas injected directly into the blood flow. A comparison of these twokinetics could be explained (quite speculatively) by a phenomenologicalmodel describing dynamic equilibrium between just two types of bindingsites (pools) of the dye in the blood-perfused heart. One pool isobviously the membranes of excitable cardiomyocytes (the “active” pool).This pool should provide voltage-sensitive fluorescence with deltaF/F˜12% as measured in the absence of blood. The second pool (the“passive” pool) is associated with blood itself (possibly includingblood cells and components of plasma). It fluoresces and increases thebackground but does not provide voltage-sensitive fluorescence, leadingto the reduction in delta F/F from 12 to 5%. Thus, when the dye isinjected in the blood flow, a significant portion of it is bound firstto the “silent” pool. Then it is slowly released from the “silent” bloodpool and is redistributed between the “passive” pool and the “active”pool. The system reaches a steady-state equilibrium yielding delta F/F˜5%. The same steady-state is apparently reached via a different route,when the dye in injected in the Tyrode's perfusion. In this case theonly pool present is the “active” pool (cardiomyocytes). The dye boundto the excitable membrane yield a signal with delta F/F=12%. When theblood is added later, the dye redistributes between the “active” pooland the “passive” pool leading to decrease in total fluorescence, OP,and delta F/F. The system reaches an equilibrium at delta F/F ˜5%.Additional experiments will be needed to substantiate these ideas.Additional experiments will be needed to provide more details on the dyeloading mechanism in blood-perfused tissue.

JPW 6003 and 6033 exhibit an excitation wavelength (˜650 nm) which wasseparated from the absorption maximum of blood (˜580 nm). This allowsuse of significantly reduced excitation power, which in our experimentscorresponded to an illumination level of <1 mW/mm² for an adequatefluorescent signal.

There is intense interest to extend these investigations to spiral andscroll waves in millimeter thick layers or across whole ventricular wall(thickness up to ˜10 mm), with the final goal being to reconstruct 3Dscroll waves. This requires new voltage sensitive dyes that exhibitexcitation/emission peaks at longer wavelengths so as to provide lowerlight scattering, and less background fluorescence from endogenouschromophores in cardiac tissue. An additional issue is to shift the dyeexcitation spectrum away from the blood absorption peak. Recently thefirst longer-wavelength (near infrared) voltage-sensitive dyes weresynthesized and have proven useful in recording cardiac and neuronalelectrical activity. New optical methods, such as transillumination,narrow illuminating beam scanning, as well as transmitted lightrecording in combination with JPW 6003 and 6033 dyes promise to improvedeeper layer imaging. Thus, in real biomedical applications, the new JPW6003 and 6033 show many advantages over the existing blue-red repertoireof voltage sensitive dyes, such as greater depth of imaging,compatibility with blood perfused samples, and prolonged retention.Differences among the JPW 6003 and 6033 dyes in terms of lipidsolubility, tissue penetrability, and ionic charge provides theopportunity to customize dyes for specific types of experimentaldemands. Additionally, optical mapping techniques for blood-perfusedcardiac tissues is important because it is more physiological andbecause it is a step towards mapping in the live animal (or human, inremote future). Blood perfusion allows studying many specific featuressuch as a stable VF baseline.

The dye JPW6003 showed delta F/F of approximately 20% in a modelmembrane, and in thick cardiac tissue specimens in transilluminationmode (˜10 mm, using excitation at 610-650 nm, emission >720 nm); and18.7% in pig tissue. In blood perfused cardiac tissue, delta F/F was 12%for JPW6003. The other dye, JPW6033, also performed very efficiently interms of loading, voltage sensitive signal, washout, and resistance tointernalization, but does not require a vehicle such as Pluronic F-127.

TABLE 1 Fluorescence efficacies and signal to noise ratio of the styryldyes JPW6003 and JPW6033 in different cardiac tissues Parameter (Dye)ΔF/F S/N ΔF/F S/N Animal (6003) (6003) (6033) (6003) Mouse  9.1 ± 1.4490 ± 50  10 ± 1.5 530 ± 55 Rat 11.1 ± 1.6 670 ± 70  14 ± 1.5 760 ± 80Guinea pig 11.3 ± 1.0 715 ± 80  12.5 ± 1.4   960 ± 95 Pig 18.7 ± 1.11000 ± 100  15 ± 1.0 850 ± 90 Pig*   20 ± 1.3  24 ± 2.2 14 ± 1.1 — Pig⁺  12 ± 1.5 — — — Pig⁺⁺  5.5 ± 1.5 — — — Values are mean ± SD. Relativefluorescence ΔF/F is expressed in percent. *transillumination ⁺bloodperfusion, Tyrode's-perfused loading ⁺⁺blood perfusion, blood-perfusedloading

Table 1 summarizes the maximal fluorescence efficacies, and maximal OPsignal to noise ratio (S/N) for the new styryl dyes in different cardiactissues. Efficacy of the new dyes (expressed as relative fluorescenceΔF/F), is significantly higher in JPW 6033 and JPW 6033 thandi-4-ANEPPS: in any of the tested species exhibited a 50-100% higher OPamplitude. The quantum efficiency of the new dyes has not been measured,but based on other similar styryl dyes we assume it to be of the orderof 0.3. Regarding tissue specific efficacy, the tendency for it toincrease as the heart gets bigger, and ventricular wall also getsthicker, i.e. progressively from mouse to pig was observed. The newstyryl dyes also provide better S/N ratio. This is usually important forthe optical mapping of cells or cell cultures. Additionally, intransillumination mode in pig the new dyes provided much higher S/Nratio than di-4-ANEPPS. The both new dyes provide practically identicalperformance, the JPW6033 being a butylsulfate analog of JPW6003 (createdfor the purpose of better loading).

TABLE 2 The washout times of the JPW6003 and JPW6033 dyes in variousspecies Dye Animal JPW6003 JPW6033 Mouse 1.4 ± 0.2 1.0 ± 0.1 Rat 2.50 ±0.28 1.8 ± 0.2 Guinea pig 2.80 ± 0.31 2.0 ± 0.2 Pig 3.33 ± 0.31 2.50 ±0.28 Values are means ± SD (in hours).

Loading agent: Specifically, many of the dyes require either a loadingagent, that may be toxic and/or change electrophysiological processes inthe cardiac tissue; or requires loading protocols that may be notphysiological, as at pH reduced to 6.0. JPW6003 to be effectively loadedwith usage of pluronic F-127; switching to pluronic L64 as suggestedelsewhere did not significantly improved the loading, but insteadincreased toxic effects. No loading agent was needed with JPW6033(Di-4-ANBDQBS).

Raw optical action potentials (OP) are quite noisy, and for furtheranalysis have been processed as described earlier. Negative polarity ofraw action potentials observed at optimal performance of the new dyesreflects fluorescence collection on the red (longer wavelength) wing ofthe dye emission spectrum. Processed signals were used for all furthermeasurement results.

All OPs are normalized in terms of delta F/F. Panel A shows OPs inTyrode-perfused tissues, and panel B in blood-perfused tissue. The JPW6033 and JPW 6003 dyes provided much higher OP amplitude (up to 3 timesin terms of delta F/F) compared to that of di-4-ANEPPS. JPW6033 alsoshowed a much higher OP amplitude than di-4-ANEPPS, and even in mostspecies higher than JPW6003, except pig (for details see Table 1).However it should be noted that a somewhat higher dye concentration(several times compared to di-4-ANEPPS) has to be applied for the newstyryl dyes to work effectively. The dye concentration of 10-100 μM(di-4-ANEPPS was applied at 10 μM) for bolus injection were found tostain tissue effectively, and not to produce any significant toxiceffect. Longer upstroke in the OP recorded by the new dyes is related todeeper sampling of the cardiac tissue due to deeper penetration of redexcitation light. There was a tendency for the OP amplitude to increaseas the heart gets bigger, and ventricular wall also gets thicker. Intransillumination mode, where much deeper layers contribute to the OPsignal, the JPW 6033 and JPW 6003 dyes most significantly outperformeddi-4-ANEPPS.

Table 1 (shown above) summarizes the maximal fluorescence efficacies(expressed as relative fluorescence ΔF/F), and maximal OP signal tonoise ratio (S/N) for the JPW 6033 and JPW 6003 dyes in differentcardiac tissues. Efficacy of the new dyes, is significantly higher thandi-4-ANEPPS: in any of the tested species exhibited a 50-100% higher OPamplitude. The quantum efficiency of the new dyes is estimated to be ofthe order of 0.3. Regarding tissue specific efficacy, there was atendency for it to increase as the heart gets bigger, and ventricularwall also gets thicker, i.e. progressively from mouse to pig. The JPW6033 and JPW 6003 dyes also provide better S/N ratio. This is usuallyimportant for the optical mapping of cells or cell cultures.Additionally, in transillumination mode in pig the new dyes providedmuch higher S/N ratio than di-4-ANEPPS. Both JPW 6033 and JPW 6003 dyesprovide practically identical performance, the JPW6033 being abutylsulfate analog of JPW6003.

In blood perfused pig tissue JPW6003 showed lower deltaF/F values butsurprisingly much better than that of di-4-ANEPPS, and comparable todi-4-ANEPPS performance in Tyrode's perfused hearts. For practicalapplications one should take into account that dye performance istransient and also depend on the loading method (see discussion).JPW6003 also was superior to di-4-ANEPPS in terms of signal-to-noiseratio.

Procedures and Dynamics of Dye Loading.

Table 2 (shown above) summarizes washout times of OP amplitude, which isan important parameter. 50% washout times (in hours) are provided formouse, rat, guinea pig and pig, respectively. The values of thisparameter progressively increase from mouse to rat to guinea pig, andpig.

Methods and Results: Absorbance and emission spectra in ethanol andmulti-lamellar vesicles (MLV), as well as voltage-dependent spectralchanges in a model lipid bilayer have been recorded. Dye performance incardiac tissue from four animal models (mouse, rat, guinea pig and pig)was examined. The dye JPW6003 showed delta F/F of approximately 20% in amodel membrane, and in thick cardiac tissue specimens intransillumination mode (˜10 mm, using excitation at 610-650 nm,emission >720 nm); and 18.7% in pig tissue. In blood perfused cardiactissue, delta F/F was 12% for JPW6003. The other dye, JPW6033, alsoperformed very efficiently in terms of loading, voltage sensitivesignal, washout, and resistance to internalization, but does not requirea vehicle such as Pluronic F-127

Experimental Section:

JPW 5020, JPW 3067, JPW 5034

JPW 5020, JPW 3067, JPW 5034 dyes were found to perform efficiently invarious cardiac tissues and to provide OP comparable to those ofdi-4-ANEPPS. The two most successful dyes, JPW3067 and JPW5034, provide40-120% of the relative fluorescence response of di-4-ANEPPS. In thinHLB layers, the new dyes provide lower relative fluorescence (5%) thandi-4-ANEPPS (9%) so that their higher efficacy in cardiac tissues isrelated to the probing of deeper layers. In transillumination mode (whenan excitation source and an emission detector are at the opposite sidesof tissue) for thick cardiac tissues (>10 mm), the relative fluorescencechange of the new dyes also approached or even exceeded that ofdi-4-ANEPPS. Optimization of filters for these dyes yet to be performedcould yield further improvement in their fluorescent efficacies.

The JPW 5020, JPW 3067, JPW 5034 dyes provide advantages in the choiceof excitation methods and/or light sources. Especially when working witha large area and/or thick cardiac tissues, one needs a quite powerfullight source capable of providing excitation at the levels of 100mW/cm2. When using a halogen light source with an excitation filter,shifting the central filter wavelength from 520 to 650 nm allows aseveral fold reduction of supplied power to the halogen bulb.Alternatively, one can use less dye or record at higher speed and stillhave good OP. The possible use of inexpensive excitation sources such asred LED, red laser diodes, and helium-neon lasers (at 633 nm) alsorepresents potential advantages. All of these sources produce highdye-excitation efficiency.

Another advantage of the JPW 5020, JPW 3067, JPW 5034 dyes is lowerendogenous absorption for both excitation and emission light in cardiactissue. Most of the endogenous fluorescence was blocked by emissionfilters; subtraction of the autofluorescence background corrected thetrue background fluorescence only about 10%. According to the opticalproperties of cardiac tissue, shifting the wavelength 150-200 nm to thered reduces the absorption coefficient several times. Measurements showthat for 10-mm-thick cardiac tissue (bloodless), this means about a30-fold increase of excitation light in deep layers. Also, becausescattering decreases with increasing wavelength, that would allow betterdepth resolution of the recorded OP. Finally, the JPW 5020, JPW 3067,JPW 5034 dyes have both excitation and emission spectra that are the farfrom the blood absorption maximum (>580 nm). This allows the possibilityof using them in blood-perfused tissues.

The dynamics of dye loading, washout, and internalization was alsostudied. This little-studied characteristic of the dyes may be importantfor long-term experiments (a half hour or longer) or large area (volume)monitoring of electrical activity in cardiac tissue. In individual cellor cell culture applications, internalization of an electrochromic dyeis the primary pathway for degradation of the OP, but in Langendorfftype or other constantly perfused preparations, dye washout also mayplay an important role. No photobleaching was observed: the dynamics ofthe fluorescent signal are the same under permanent or intermittent(several seconds each minute) excitation.

Although there were generally no significant differences between theloading times measured using the three parameters, the washout timesgenerally increase in the order OP background fluorescence relativepotentiometric fluorescence change (delta F/F). This would be thebehavior expected if dye internalization was the main factor degradingthe efficacy of the potentiometric probes over time. The values of allthree parameters are lowest for the dye JPW3067 and gradually increasefor JPW5034 and JPW5020. This is related to the gradually increasingmolecular weight (and also size) as well as the length of thehydrocarbon chains of the dye molecules. Longer hydrocarbon chains makemore hydrophobic dyes that bind to a cell membrane more tightly andinternalize more slowly, according to systematic studies with the ANEPdye series. During washout, the OP amplitude decreases faster than thetotal fluorescence, suggesting that some dye molecules are beinginternalized. The relatively fast loading and washout of JPW3067 can bequite an advantage in specific applications. Considering thespecies-specific dynamics, the dye loading and washout progressivelyslows down from rat to guinea pig to pig. Although metabolic ratefollows a similar trend (being highest in the rat and lowest in thepig), it remains to be determined whether metabolic rate influences thewashout dynamics of these dyes.

While particular embodiments of the present invention have beendescribed herein for purposes of illustration, many modifications andchanges will become apparent to those skilled in the art. Variousmodifications and variations of the described apparatus, kit, method,and system of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific embodimentsoutlined above, it should be understood that the invention should not beunduly limited to such specific embodiments. Accordingly, the appendedclaims are intended to encompass all such modifications and changes asfall within the true spirit and scope of this invention.

We claim:
 1. An optically mapping composition comprising avoltage-sensitive dye of the formula:

wherein R¹ is a first hydrocarbon group that is a butyl group; R² is asecond hydrocarbon group that is a butyl group; and R³ is a butylsulfonate polar group.
 2. The composition of claim 1, further comprisinga delivery agent that is at least one of a physiologically acceptableadjuvant, solvent, substrate, or diluent.
 3. The composition of claim 2,wherein the delivery agent is selected from the group consisting of:Pluronic F-127, Pluronic L64, cyclodextrin, gamma cyclodextrin,dimethylsulfoxide, ethanol, and combinations thereof.